Researchers from the Institute for Biological and Medical Imaging at Helmholtz Zentrum München and Technische Universität München hypothesized that an acoustic cavity could improve the performance of a π-phase-shifted fibre Bragg grating (π-FBG) detector to enable lower-cost O2A intravital microscopy (Optica 4 1180).

The miniaturized sensor developed by Rami Shnaiderman and co-authors consists of an ellipsoidal acoustic cavity, a metal sleeve adaptor that fits the acoustic cavity to a microscope objective and the π-FBG ultrasound detector. The sensor has several unique features that enable its intravital epi-illumination performance. Its flexible design allows adaptation to work with wide variety of optical microscopes using the simple metal sleeve adapter. Signal amplification made possible by the acoustic cavity improves the signal-to-noise ratio (SNR) and enables use of simple continuous-wave laser interrogation of the π-FBG detector. This feature improves robustness, reduces form factor over prior sensor designs and reduces cost by a factor of 10.

The ellipsoidal shape of the acoustic cavity gives rise to two acoustic focuses: an external focus positioned inside the sample that coincides with the optical focus, and an internal focus that coincides with the π-FBG detector. The external acoustic focus, engineered to be 400 µm from the rim of the cavity, defines the maximal imaging depth, suitable for the study of mouse dermis. It also matches the depths commonly imaged by multi-photon microscopy. The internal acoustic focus is 5 mm away from the external, along the major axis of the ellipse. This configuration creates an acoustic path that is different from the optical path, eliminating all interference between the microscope's optical path and the π-FBG detector.

The cavity's dimensions – 9.6 mm along the major axis and 8.2 mm along the minor axis – were chosen to maximize the solid angle of the sensor and minimize ultrasound attenuation by minimizing the acoustic propagation path. Any detected signal is amplified five times by the cavity's ability to collect and focus ultrasound waves onto the π-FBG detector. The cavity is filled with centrifuged ultrasound gel to allow for acoustic impedance matching. The sensor's field-of-view (FOV) can be expanded to at least 0.63 x 0.63 mm, the result of the large aperture of the acoustic cavity. When combined with galvanometric mirrors, this large FOV enables fast scanning speeds.

In vivo demonstration

The authors explain that the sensor operates when laser beams for second-harmonic generation (SHG) and optoacoustic excitation leave the objective and enter the acoustic cavity through an opening, focusing inside the specimen. The excitation beams scan the region of interest. Reflected optical signals are collected by the microscope objective and ultrasonic signals enter the acoustic cavity, where they are reflected and focused onto the active detection area of the π-FBG detector.

The team successfully conducted in vivo imaging of the thin tissue of a mouse's ear and thick tissue of the abdominal dermis of a mouse, by stitching together scans over areas of 2 x 2 mm and 1.8 x 2 mm, respectively. They reported that the vasculature could be seen in good detail down to the smallest capillaries.

The optoacoustic images were complemented by SHG imaging of collagen structures in the epidermis. "Optoacoustic contrast reveals structured and functional variations in microvasculature associated with the diameters and density of blood vessels, and can enhance the information available to studies of hypertension, obesity, diabetes, inflammation or angiogenesis," they wrote.

"Optoacoustic imaging is a modality that visualizes absorption contrast. This powerful technique provides important information on the structural and functional variations of absorbers, most notably on diameter, densities and oxygenation of vasculature. In addition, ultrasound attenuation in tissue is negligible compared to that of light, enabling enhanced imaging depths," Shnaiderman told medicalphysicsweb.

He continued: "Optoacoustic imaging has shown great success in non-invasive diagnosis of sentinel lymph glands in melanoma, human carotid atheroma and thyroid nodules. I believe our sensor will accelerate the study of many more medical conditions and biological processes by upgrading the standard optical microscopes readily available in any lab, without hindering their daily function or requiring operation by specialized personnel."

"We strive to increase the performance of future sensors and reduce complexity and cost to promote widespread implementation. We are confident that optoacoustic microscopy will be an invaluable add-on to any microscope, boosting medical and fundamental research," Shnaiderman concluded.